Project Summary/Abstract Exploration of new agents and combination therapies, targeting novel pathways associated with tumor proliferation, invasion, angiogenesis and apoptosis resistance will lead to improved patient outcomes in lung cancer. In fact, targeted therapy with an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) has been shown to prolong survival in advanced non-small cell lung cancer (NSCLC). How can we improve these therapies? An understanding of the mechanisms of action of targeted agents will help us define the mechanisms of resistance. Signaling through the EGFR and cyclooxygenase-2 (COX-2) involves pathways that interact to promote the malignant phenotype. Our pre-clinical and clinical studies support these findings and we have identified novel signaling events that implicate tumor COX-2 expression as a contributor to resistance to EGFR inhibitors in NSCLC. Very recent clinical trials strongly support continued investigation of targeting both pathways in NSCLC. In laboratory-based studies conducted in this project we discovered novel mechanisms of resistance to EGFR TKI in NSCLC; this resistance is mediated through an EGFR-independent activation of the MAPK/Erk signaling pathway by the COX-2 metabolite PGE2. Thus, more effective therapy may require blocking both the EGFR-dependent as well as EGFR-independent pathways to MAPK/Erk activation in NSCLC. Based on our laboratory findings, we conducted a phase I clinical trial evaluating the combination of erlotinib and celecoxib in advanced NSCLC. The results of the phase I study: 1) established the optimal biological dose of celecoxib based on urine PGE-M measurements and 2) showed no unexpected toxicity. We hypothesize that: 1) PGE2-dependent activation of MAPK/Erk signaling serves to activate downstream signaling pathways leading to tumor proliferation despite the presence of an EGFR TKI, 2) analysis of the pathways responsible for the PGE2-dependent activation of MAPK/Erk will help identify additional therapeutic targets, 3) COX-2-regulated proteins play a role in erlotinib resistance, and, therefore, 4) dual inhibition of the EGFR and COX-2 pathways will result in changes in associated proteins that can subsequently be utilized for response prediction and patient selection for this combination therapy. The studies proposed in Aims 1 - 4 will determine the signaling mechanisms responsible for PGE2-mediated, EGFR- independent activation of the MAPK/Erk pathway in NSCLC and identify additional therapeutic targets. We will also define targets that can be utilized as markers of resistance/sensitivity to this combined therapy. Aim 5 seeks to determine COX-2-dependent proteins associated with resistance or sensitivity to erlotinib / celecoxib therapy in specimens from an ongoing Phase II clinical trial, including pre- and post-therapy tumor biopsies and plasma samples. This will allow identification of proteins that can be readily assessed as markers of response and / or for patient selection. The data obtained in the proposed study should provide the necessary evidence to prospectively utilize tumor COX-2 and/or downstream makers of pathway activity as selection biomarkers in future studies. PUBLIC HEALTH RELEVANCE: Relevance to Veterans Health. Successful therapy for lung cancer remains an unattained goal for many of our VA patients. Each year lung cancer exacts a devastating toll in terms of lives lost and resources expended. It is expected that this VA supported research will ultimately lead to improvement in disease-free survival for veterans with lung cancer.